JP2017148004A - Nucleic acid amplification reaction apparatus and nucleic acid amplification reaction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification reaction apparatus capable of accelerating PCR.SOLUTION: The present invention provides a nucleic acid amplification reaction apparatus comprising an attachment part capable of attaching a reaction container in which a reaction liquid is arranged in a flow pass, a temperature gradient forming part for forming a temperature gradient in the longitudinal direction of the flow pass, and a movement mechanism that moves the attachment part in the direction including the longitudinal component and moves the reaction liquid relatively in the longitudinal direction of the flow pass.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a nucleic acid amplification reaction apparatus and a nucleic acid amplification reaction method.

  In recent years, gene-based medical care such as gene diagnosis and gene therapy has attracted attention due to the development of gene utilization technology, and many methods using genes for variety discrimination and variety improvement have been developed in the field of agriculture and livestock. . As a technique for utilizing a gene, a technique such as a PCR (Polymerase Chain Reaction) method is widely used. Today, PCR has become an indispensable technique for elucidating information on biological materials.

  The PCR method is a method of amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid (target nucleic acid) to be amplified and a reagent to thermal cycling. The thermal cycle is a process in which two or more stages of temperature are periodically applied to the reaction solution. In the PCR method, a method of applying a two-stage or three-stage thermal cycle is common.

  For example, in Patent Document 1, a reaction vessel filled with reaction droplets and a liquid (oil or the like) that is immiscible with the reaction droplets and has a specific gravity smaller than that of the reaction solution is rotated around the rotation axis, and the specific gravity is increased. A nucleic acid amplification reaction apparatus is described in which a reaction solution is moved by the difference and thermal cycling is performed.

JP 2012-115208 A

  However, in the nucleic acid amplification reaction apparatus described in Patent Document 1, since the reaction solution is moved by gravity, the moving speed of the reaction solution is limited, and it may be difficult to increase the PCR speed.

  One of the objects according to some aspects of the present invention is to provide a nucleic acid amplification reaction apparatus capable of increasing the speed of PCR. Another object of some embodiments of the present invention is to provide a nucleic acid amplification reaction method capable of increasing the speed of PCR.

The nucleic acid amplification reaction apparatus according to the present invention comprises:
A mounting portion on which a reaction vessel in which a reaction solution is arranged in a flow path can be mounted;
A temperature gradient forming section for forming a temperature gradient in the longitudinal direction of the flow path;
A moving mechanism for moving the mounting portion in a direction including the component in the longitudinal direction and relatively moving the reaction solution in the longitudinal direction of the flow path;
including.

  In such a nucleic acid amplification reaction apparatus, for example, compared with the case where PCR is performed by moving the reaction solution only by gravity, the moving part can be moved faster by the moving mechanism, and the moving speed of the reaction solution can be increased. can do. Therefore, in such a nucleic acid amplification reaction apparatus, the speed of PCR can be increased.

In the nucleic acid amplification reaction apparatus according to the present invention,
The moving mechanism may swing the mounting portion.

  In such a nucleic acid amplification reaction apparatus, the reaction solution can be reciprocated in the longitudinal direction of the flow path in which the temperature gradient is formed, and the reaction solution can be subjected to a temperature cycle.

In the nucleic acid amplification reaction apparatus according to the present invention,
The moving mechanism may move the mounting portion in the longitudinal direction.

  In such a nucleic acid amplification reaction apparatus, the reaction solution can be moved more reliably in the longitudinal direction of the flow path in which the temperature gradient is formed.

In the nucleic acid amplification reaction apparatus according to the present invention,
The longitudinal direction may be a horizontal direction.

  In such a nucleic acid amplification reaction apparatus, the gravity acting on the reaction solution is constant while the reaction solution is subjected to a temperature cycle. Therefore, adjustment of the moving speed of the mounting part by the moving mechanism (setting of the moving speed) It can be done easily.

In the nucleic acid amplification reaction apparatus according to the present invention,
The longitudinal direction is a direction inclined with respect to the horizontal direction,
The flow path is
A first temperature region and a second temperature region having a temperature lower than that of the first temperature region;
The second temperature region may be located below the first temperature region in a direction in which gravity acts.

  In such a nucleic acid amplification reaction apparatus, the movement of the reaction solution can be suppressed by gravity when performing fluorescence measurement of the reaction solution located in the second temperature region. Thereby, in the nucleic acid amplification reaction apparatus, the variation in the luminance of the fluorescence can be reduced.

In the nucleic acid amplification reaction apparatus according to the present invention,
The reaction vessel may be filled with a liquid that is immiscible with the reaction solution.

  In such a nucleic acid amplification reaction apparatus, the reaction solution can be made into droplets.

In the nucleic acid amplification reaction apparatus according to the present invention,
The moving mechanism may impart acceleration to the reaction liquid by changing a moving speed of the mounting portion in a direction including the longitudinal component.

  In such a nucleic acid amplification reaction apparatus, the reaction solution can be moved relative to the flow path by acceleration.

In the nucleic acid amplification reaction apparatus according to the present invention,
The moving mechanism may impart an acceleration larger than a gravitational acceleration to the reaction solution.

  In such a nucleic acid amplification reaction apparatus, the speed of PCR can be further increased.

The nucleic acid amplification reaction method according to the present invention comprises:
Forming a temperature gradient in the longitudinal direction of the flow path in which the reaction solution is disposed;
Moving the reaction vessel having the flow path in a direction including the component in the longitudinal direction, and moving the reaction liquid relatively in the longitudinal direction of the flow path;
including.

  In such a nucleic acid amplification reaction method, for example, the moving speed of the reaction solution can be increased compared to the case where PCR is performed by moving the reaction solution only by gravity. Therefore, in such a nucleic acid amplification reaction method, the speed of PCR can be increased.

Sectional drawing which shows typically the nucleic acid amplification reaction apparatus which concerns on this embodiment. The schematic diagram for demonstrating the nucleic acid amplification reaction method using the nucleic acid amplification reaction apparatus which concerns on this embodiment. The flowchart for demonstrating the nucleic acid amplification reaction method using the nucleic acid amplification reaction apparatus which concerns on this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction apparatus which concerns on the 1st modification of this embodiment. Sectional drawing which shows typically the nucleic acid amplification reaction apparatus which concerns on the 2nd modification of this embodiment.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.

1. Nucleic acid amplification reaction apparatus 1.1. Configuration First, the nucleic acid amplification reaction apparatus according to the present embodiment will be described with reference to the drawings. FIG. 1 is a cross-sectional view schematically showing a nucleic acid amplification reaction apparatus 100 according to this embodiment. In FIG. 1, the direction in which gravity acts (hereinafter also referred to as “gravity acting direction”), the downward direction is indicated by an arrow g, and the horizontal direction perpendicular to the gravity acting direction is indicated by an arrow h.

  As shown in FIG. 1, the nucleic acid amplification reaction apparatus 100 includes heating units 20 and 22 to which the reaction vessel 10 can be attached, a fluorescence measurement unit 30, a support unit 40, a moving mechanism 50, and a control unit 60. Including. FIG. 1 shows a state in which the reaction vessel 10 is fixed to the heating units 20 and 22. For convenience, FIG. 1 shows the moving mechanism 50 and the control unit 60 as functional blocks.

  The reaction vessel 10 has a flow path 12. The channel 12 is provided in a straight line from the bottom 14 to the lid 16 of the reaction vessel 10. For example, the channel 12 has a longitudinal direction L that is parallel to a straight line connecting the center of the bottom 14 and the center of the lid 16. In the illustrated example, the longitudinal direction L is the horizontal direction h (parallel to the horizontal direction h).

  The reaction liquid 4 and the liquid 6 are disposed in the flow path 12. In the illustrated example, the flow path 12 is filled (filled) with the reaction solution 4 and the liquid 6. The reaction liquid 4 is held in the liquid 6 in the form of droplets. In the illustrated example, the shape of the reaction solution 4 is a sphere. The reaction liquid 4 has a higher specific gravity than the liquid 6. As the reaction vessel 10 moves, the reaction solution 4 moves in the flow path 12 relative to the reaction vessel 10. The reaction solution 4 includes, for example, DNA (target nucleic acid) amplified by PCR, an enzyme such as DNA polymerase necessary for amplifying the DNA, a primer, a fluorescent probe for detecting a nucleic acid amplification product, and the like.

The volume of the reaction solution 4 is, for example, 0.1 μL or more and 5 μL or less, and preferably about 1 μL. By setting the volume of the reaction liquid 4 to 0.1 μL or more, the ratio of the surface area to the mass of the reaction liquid 4 can be suppressed, and consequently the frictional resistance with the liquid 6 can be suppressed. It can move smoothly. Therefore, it becomes possible to control the reaction solution 4 to a desired temperature. On the other hand, when the volume of the reaction solution 4 is 5 μL or less, the time required for the temperature change of the reaction solution 4 can be kept short, and the speeding up of PCR can be realized. Further, it is not necessary to increase the inner diameter of the reaction vessel 10 more than necessary, which contributes to downsizing of the apparatus.

  The liquid 6 is a liquid that is not miscible with the reaction liquid 4, that is, does not mix. The liquid 6 has a specific gravity smaller than that of the reaction liquid 4. The liquid 6 is, for example, dimethyl silicone oil or paraffin oil.

  The liquid 6 may not be disposed in the flow path 12. That is, the liquid 6 may not be present in the reaction vessel 10. In this case, the reaction solution 4 is not in the form of droplets, but moves relative to the reaction vessel 10 through the flow path 12 in, for example, an irregular shape.

  The material of the reaction vessel 10 is, for example, glass, polymer, metal or the like. The inner diameter of the reaction vessel 10 is, for example, 2 mm or more and 3 mm or less. In the illustrated example, the bottom portion 14 of the reaction vessel 10 has a bottom surface that is a parabolic surface.

  The first heating unit 20 and the second heating unit 22 are members that transmit heat generated from a heater (not shown) to the flow path 12. The heating units 20 and 22 are in contact with the reaction vessel 10, for example. The heating units 20 and 22 are provided to be separated from each other, for example. The heating units 20 and 22 are provided side by side along the longitudinal direction of the flow path 12. The reaction vessel 10 is fixed to the heating units 20 and 22.

  The first heating unit 20 and the second heating unit 22 are, for example, aluminum heat blocks. By making the heat block made of aluminum, the reaction vessel 10 can be efficiently heated.

  The first heating unit 20 is provided with a first opening 21. The channel 12 has a first temperature region 12a. The first temperature region 12 a is a portion located in the first opening 21 of the flow path 12. The first opening 21 is a through hole that penetrates the first heating unit 20. In the illustrated example, a part of the lid 16 is located in the first opening 21.

  The first heating unit 20 can heat the first temperature region 12a to the first temperature. Specifically, the first heating unit 20 can heat the reaction solution 4 and the liquid 6 located in the first temperature region 12a to the first temperature. The first temperature is, for example, 90 ° C. or higher and 110 ° C. or lower, and preferably 105 ° C. The first temperature is a temperature suitable for denaturation (dissociation of double-stranded DNA) in PCR.

  The second heating unit 22 is provided with a second opening 23. The flow path 12 has a second temperature region 12b different from the first temperature region 12a. The second temperature region 12 b is a portion located in the second opening 23 of the flow path 12. The second opening 23 is a through hole that penetrates the second heating unit 22. In the illustrated example, the bottom 14 is located in the second opening 23. Since the second opening 23 is a through hole, the fluorescence measuring unit 30 can perform fluorescence measurement by irradiating the second temperature region 12b with light.

  Although not shown, when the fluorescence measurement unit 30 does not perform fluorescence measurement, the second opening 23 may be a hole with a bottom, and the bottom 14 may be covered with the second heating unit 22. Thereby, the contact area of the reaction container 10 and the 2nd heating part 22 can be increased, and the 2nd heating part 22 can heat the 2nd temperature area | region 12b to more desired temperature.

The second heating unit 22 can heat the second temperature region 12b to the second temperature. Specifically, the second heating unit 22 can heat the reaction solution 4 and the liquid 6 located in the second temperature region 12b to the second temperature. The second temperature is a temperature different from the first temperature. Specifically, the second temperature is lower than the first temperature, for example, 50 ° C. or higher and 70 ° C. or lower, and preferably 54 ° C. Therefore, the temperature of the second temperature region 12b is lower than that of the first temperature region 12a. The second temperature is a temperature suitable for annealing (a reaction in which a primer binds to a single-stranded DNA) and an extension reaction (a reaction in which a complementary strand of DNA is formed starting from a primer) in PCR.

  The temperatures of the first heating unit 20 and the second heating unit 22 may be controlled by a temperature sensor and control unit 60 (not shown). The heating units 20 and 22 provided with the openings 21 and 23 are mounting units to which the reaction vessel 10 can be mounted. Since the first temperature and the second temperature are different from each other as described above, the heating units 20 and 22 are temperature gradient forming units that form a temperature gradient in the longitudinal direction of the flow path 12. As described above, when the first temperature is higher than the second temperature, a temperature gradient in which the temperature gradually decreases from the first temperature region 12a toward the second temperature region 12b is formed in the flow path 12. The

  The fluorescence measuring unit 30 irradiates the reaction solution 4 with light, and detects fluorescence emitted from the reaction solution 4 (fluorescence of the reaction solution 4). The fluorescence measuring unit 30 is provided at a position facing the bottom 14 of the reaction vessel 10. The material of the reaction vessel 10 is a material that can transmit the light emitted from the fluorescence measurement unit 30 and the fluorescence of the reaction solution 4. The fluorescence measuring unit 30 can irradiate the reaction solution 4 with light, for example, when the reaction solution 4 is in contact with the bottom 14. The fluorescence measurement unit 30 may continue to emit light while performing PCR.

  The support unit 40 supports the heating units 20 and 22 and the fluorescence measurement unit 30. The heating units 20 and 22 and the fluorescence measurement unit 30 are fixed to the support unit 40. The support portion 40 includes a first portion 42, a second portion 44, and a third portion 46. The first portion 42 supports the fluorescence measurement unit 30. The shape of the first portion 42 is, for example, a rectangular parallelepiped. The second portion 44 and the third portion 46 sandwich and support the heating units 20 and 22 and the first portion 42. The shape of the 2nd part 44 and the 3rd part 46 is plate shape, for example. In addition, the shape of the support part 40 will not be specifically limited if the heating parts 20 and 22 and the fluorescence measurement part 30 can be supported. Further, the material of the support portion 40 is not particularly limited.

The movement mechanism 50 is a mechanism that moves the support unit 40 under the control of the control unit 60. The moving mechanism 50 moves the reaction vessel 10, the heating units 20 and 22, and the fluorescence measuring unit 30 in a direction including a component in the longitudinal direction L (in the illustrated example, in the longitudinal direction L) by moving the support unit 40. Thus, the reaction solution 4 is moved relative to the reaction vessel 10 (relative to the flow channel 12) in the longitudinal direction L of the flow channel 12. For example, the moving mechanism 50 swings (vibrates, reciprocates) the support unit 40 (the reaction vessel 10, the heating units 20, 22 and the fluorescence measurement unit 30). Thereby, the reaction vessel 10 reciprocates along the longitudinal direction L, and the reaction solution 4 reciprocates between the first temperature region 12 a and the second temperature region 12 b of the flow path 12. The movement of the reaction solution 4 is described in “1.2.
This will be described in detail in “Nucleic acid amplification reaction method”. For example, the moving mechanism 50 changes the moving speed (of the heating units 20 and 22) of the support unit 40 in the direction including the component in the longitudinal direction L to apply acceleration to the reaction liquid 4. The moving mechanism 50 may give the reaction liquid 4 an acceleration larger than the gravitational acceleration.

  As the moving mechanism 50, for example, a member provided with a clamping part (not shown) that clamps the support part 40 and a motor (not shown) that vibrates the clamping part is used. The period of vibration of the support portion 40 by the moving mechanism 50 may be constant or may not be constant. The form of the moving mechanism 50 is not particularly limited as long as the support unit 40 can be moved to move the reaction solution 4 relative to the reaction vessel 10.

The control unit 60 controls the moving mechanism 50. The control unit 60 is, for example, a CPU (Centr
al Processing Unit) and a storage unit (ROM (Read Only Memory), RAM (Random Access Memory)). In the storage unit, for example, various programs and data for controlling the moving mechanism 50 are recorded. For example, the storage unit temporarily records data, processing results, and the like during various processes performed by the control unit 60.

  The detection result of the fluorescence measurement unit 30 may be stored in the recording unit of the control unit 60. The control unit 60 can acquire, for example, a PCR nucleic acid amplification curve based on the fluorescence brightness (fluorescence brightness) detected by the fluorescence measurement unit 30, and determine the number of PCR temperature cycles.

1.2. Nucleic Acid Amplification Reaction Method Next, a nucleic acid amplification reaction method according to this embodiment will be described with reference to the drawings. Hereinafter, a nucleic acid amplification reaction method using the nucleic acid amplification reaction apparatus 100 will be described. FIG. 2 is a schematic diagram for explaining a nucleic acid amplification reaction method using the nucleic acid amplification reaction apparatus 100. FIG. 3 is a flowchart for explaining a nucleic acid amplification reaction method using the nucleic acid amplification reaction apparatus 100. For convenience, illustration of the moving mechanism 50 and the control unit 60 is omitted in FIG. 2.

  First, the reaction vessel 10 filled with the reaction solution 4 and the liquid 6 is fixed to the heating units 20 and 22 (step S102). Specifically, the reaction vessel 10 is fixed to the heating units 20 and 22 by being inserted into the openings 21 and 23. For example, the reaction liquid 4 and the liquid 6 are injected into the reaction container 10 and then filled into the reaction container 10 by tightening the lid 16. Each of the heating units 20 and 22 may be formed of a heat block divided into two, and the reaction vessel 10 may be fixed to the heating units 20 and 22 by being sandwiched between the heat blocks.

  In step S102, the reaction solution 4 is located in the first temperature region 12a of the flow path 12, as shown in FIG. In the example shown in FIG. 2A, the reaction solution 4 is disposed in contact with the lid portion 16.

  Next, the reaction vessel 10 is heated by the heating units 20 and 22, and a temperature gradient is formed in the longitudinal direction L of the flow path 12 (step S104). Specifically, the first heating unit 20 heats the first temperature region 12a of the flow path 12 to the first temperature. The second heating unit 22 heats the second temperature region 12b of the flow path 12 to the second temperature. As a result, a temperature gradient is formed in which the temperature decreases from the first temperature region 12a toward the second temperature region 12b.

  In step S104, the reaction solution 4 is located in the first temperature region 12a as shown in FIG. Accordingly, the reaction solution 4 is heated to the first temperature, and the reaction (denaturation) at the first temperature is performed on the reaction solution 4.

Next, the control unit 60 controls the moving mechanism 50 to move the reaction solution 4 from the first temperature region 12a to the second temperature region 12b (step S106). Specifically, the control unit 60 controls the moving mechanism 50 to move the support unit 40 (the reaction vessel 10) in the longitudinal direction L (in the illustrated example, one direction L1 of the longitudinal direction L) (see FIG. The state shown in FIG. 2A is changed to the state shown in FIG. Thereafter, the control unit 60 controls the moving mechanism 50 to stop the movement of the support unit 40 (from the state shown in FIG. 2B to the state shown in FIG. 2C). That is, the control unit 60 controls the moving mechanism 50 to give the reaction liquid 4 an acceleration in the direction opposite to the moving direction of the support unit 40. When the movement of the support 40 is stopped or decelerated, an inertial force acts on the reaction solution 4, and the reaction solution 4 moves relative to the reaction vessel 10 (relative to the flow path 12) in the longitudinal direction L. Thus, the first temperature region 12a moves to the second temperature region 12b. In the example shown in FIG. 2C, the reaction solution 4 is disposed in contact with the bottom portion 14. The moving mechanism 50 may give the reaction liquid 4 an acceleration larger than the gravitational acceleration when moving the reaction liquid 4 from the first temperature region 12a to the second temperature region 12b.

  In this embodiment, when step S106 is performed subsequent to step S104, that is, when the first step S106 is performed, the control unit 60 uses a temperature sensor (not shown) (the temperature connected to the first heating unit 20). When it is determined that a predetermined time has elapsed after the sensor) reaches a predetermined temperature, the reaction solution 4 is moved from the first temperature region 12a to the second temperature region 12b.

  While the reaction liquid 4 is located in the second temperature region 12b, the reaction liquid 4 is heated to the second temperature, and the reaction at the second temperature (annealing and extension reaction) is performed on the reaction liquid 4.

  Next, the control unit 60 determines whether or not the fluorescence luminance detected by the fluorescence measurement unit 30 is greater than or equal to a predetermined value (step S108). Specifically, as shown in FIG. 2C, the control unit 60 detects the fluorescence brightness (reaction) detected by the fluorescence measurement unit 30 while the reaction solution 4 is located in the second temperature region 12b. The fluorescence brightness of the liquid 4 is acquired. And the control part 60 determines whether the acquired fluorescence brightness is more than the predetermined value previously memorize | stored in the memory | storage part.

  In step S108, when the control unit 60 determines that the detected fluorescence luminance is equal to or higher than a predetermined value (Yes), the control unit 60 stops driving the moving mechanism 50 and completes the processing (END). If the control unit 60 determines that the detected fluorescence brightness is less than the predetermined value (No), the process proceeds to step S110.

  If No in step S108, the control unit 60 controls the moving mechanism 50 to move the reaction solution 4 from the second temperature region 12b to the first temperature region 12a (step S106). Specifically, the control unit 60 controls the moving mechanism 50 to move the support unit 40 in the longitudinal direction L (in the illustrated example, the other direction L2 of the longitudinal direction L) (see FIG. 2C). The state shown in FIG. 2 is changed to the state shown in FIG. Thereafter, the control unit 60 controls the moving mechanism 50 to stop the movement of the support unit 40 (from the state shown in FIG. 2D to the state shown in FIG. 2A). That is, the control unit 60 controls the moving mechanism 50 to give the reaction liquid 4 an acceleration in the direction opposite to the moving direction of the support unit 40. When the movement of the support portion 40 is stopped or decelerated, an inertial force acts on the reaction solution 4, and the reaction solution 4 moves relative to the reaction vessel 10 in the longitudinal direction L to move from the second temperature region 12 b. Move to the first temperature region 12a. The moving mechanism 50 may give the reaction liquid 4 an acceleration larger than the gravitational acceleration when moving the reaction liquid 4 from the second temperature region 12b to the first temperature region 12a.

  Next, step S106 is started again. Then, the control unit 60 swings the support unit 40 at a constant period until it is determined in step S108 that the fluorescence luminance is equal to or higher than a predetermined value (Yes). For example, PCR can be completed by performing this temperature cycle 50 times (50 cycles) or more.

  When the reaction solution 4 moves from the first temperature region 12a to the second temperature region 12b and further moves from the second temperature region 12b to the first temperature region 12a, the temperature cycle is counted as one rotation. For example, in one rotation of the temperature cycle, the reaction solution 4 is located in the first temperature region 12a for about 1 second and is located in the second temperature region 12b for about 2 seconds.

In addition, the first temperature, the second temperature, the period of the support unit 40, the size of the heating units 20 and 22, and the like take into account the type and amount of the reaction solution 4 and the liquid 6, the size of the reaction vessel 10, and the like. Determined as appropriate,
The nucleic acid amplification reaction apparatus 100 has the following features, for example.

  In the nucleic acid amplification reaction apparatus 100, the heating units 20 and 22 are moved in the direction including the components in the longitudinal direction L of the flow path 12, and the reaction solution 4 is moved in the longitudinal direction with respect to the reaction vessel 10 (relative to the flow path 12). A moving mechanism 50 that moves relative to L is included. Therefore, in the nucleic acid amplification reaction apparatus 100, the speed at which the heating units 20 and 22 are moved by the moving mechanism 50 can be increased compared to the case where PCR is performed by moving the reaction solution 4 only by gravity, for example. The moving speed of 4 can be increased. Therefore, the nucleic acid amplification reaction apparatus 100 can increase the speed of PCR.

  In the nucleic acid amplification reaction apparatus 100, the moving mechanism 50 swings the heating units 20 and 22. Therefore, in the nucleic acid amplification reaction apparatus 100, the reaction solution 4 can be reciprocated in the longitudinal direction L of the flow path 12 where the temperature gradient is formed, and the reaction solution 4 can be subjected to a temperature cycle.

  In the nucleic acid amplification reaction apparatus 100, the moving mechanism 50 moves the heating units 20 and 22 in the longitudinal direction L. Therefore, in the nucleic acid amplification reaction apparatus 100, the reaction solution 4 can be moved more reliably in the longitudinal direction L of the flow path 12 where the temperature gradient is formed.

  In the nucleic acid amplification reaction apparatus 100, the longitudinal direction L is a horizontal direction. Therefore, in the nucleic acid amplification reaction apparatus 100, the gravity acting on the reaction solution 4 is constant while the reaction solution 4 is subjected to the temperature cycle. Therefore, the moving mechanism 50 adjusts (moves) the moving speed of the heating units 20 and 22. Speed setting) can be easily performed. Furthermore, when the longitudinal direction L is a direction inclined with respect to the horizontal direction, there may be a problem that the reaction solution 4 is brought into close contact with the bottom portion 14 due to gravity and the reaction solution 4 is difficult to separate from the bottom portion 14. In the amplification reaction apparatus 100, such a problem can be avoided.

  In the nucleic acid amplification reaction apparatus 100, the reaction vessel 10 is filled with a liquid 6 that is immiscible with the reaction solution 4. Therefore, in the nucleic acid amplification reaction apparatus 100, the reaction solution 4 can be made into a droplet state.

  In the nucleic acid amplification reaction apparatus 100, the moving mechanism 50 changes the moving speed of the heating units 20 and 22 in the direction including the component in the longitudinal direction L to give acceleration to the reaction solution 4. Therefore, in the nucleic acid amplification reaction apparatus 100, the reaction solution 4 can be moved relative to the flow path 12 by acceleration.

  In the nucleic acid amplification reaction apparatus 100, the moving mechanism 50 gives the reaction solution 4 an acceleration larger than the gravitational acceleration. Therefore, the nucleic acid amplification reaction apparatus 100 can further increase the speed of PCR.

  Although not shown, the nucleic acid amplification reaction apparatus 100 may include a third heating unit that can heat the flow path 12 to a third temperature different from the first temperature and the second temperature. The third temperature is, for example, a temperature lower than the first temperature and higher than the second temperature. Thereby, 2nd temperature can be made into the temperature suitable for annealing, and 3rd temperature can be made into the temperature suitable for extension reaction. Thus, annealing and extension reaction can be performed at different temperatures, and PCR can be optimized. Further, the number of heating units may be four or more.

2. 2. Modification of nucleic acid amplification reaction apparatus 2.1. First Modification Next, a nucleic acid amplification reaction apparatus according to a first modification of the present embodiment will be described with reference to the drawings. FIG. 4 is a cross-sectional view schematically showing a nucleic acid amplification reaction apparatus 200 according to the first modification of the present embodiment. In FIG. 4 and FIG. 5 shown below, the gravitational action direction, the downward direction is indicated by an arrow g, and the horizontal direction orthogonal to the gravitational action direction is indicated by an arrow h. For convenience, in FIG. 4 and FIG. 5 described below, the moving mechanism 50 and the control unit 60 are shown as functional blocks.

  Hereinafter, in the nucleic acid amplification reaction apparatus 200 according to the first modification of the present embodiment, members having the same functions as those of the constituent members of the nucleic acid amplification reaction apparatus 100 according to the present embodiment described above are denoted by the same reference numerals, Detailed description thereof is omitted. The same applies to the nucleic acid amplification reaction apparatus according to the second modification of the present embodiment described below.

  In the nucleic acid amplification reaction apparatus 100 described above, the longitudinal direction L of the flow channel 12 is the horizontal direction h, as shown in FIG. In contrast, in the nucleic acid amplification reaction apparatus 200, as shown in FIG. 4, the longitudinal direction L of the flow path 12 is not the horizontal direction h. The longitudinal direction L is not particularly limited, and may be, for example, a vertical direction orthogonal to the horizontal direction h. However, in the illustrated example, the longitudinal direction L is a direction inclined with respect to the horizontal direction h. The second temperature region 12b is located below in the gravitational action direction g than the first temperature region 12a.

  The inclination angle θ of the longitudinal direction L with respect to the horizontal direction h is, for example, 3 ° to 45 °, and preferably 5 ° to 15 °. When the inclination angle θ is smaller than 3 °, the reaction solution 4 may move when measuring the fluorescence of the reaction solution 4. When the inclination angle θ is greater than 45 ° C., the gravity acting on the reaction solution 4 increases, and it may be difficult to move the reaction solution 4 to the first temperature region 12a.

  In the nucleic acid amplification reaction apparatus 200, the longitudinal direction L is a direction inclined with respect to the horizontal direction h, and the second temperature region 12b is located below in the gravitational action direction g than the first temperature region 12a. . Therefore, in the nucleic acid amplification reaction apparatus 200, when the fluorescence measurement of the reaction solution 4 located in the second temperature region 12b is performed, the movement of the reaction solution 4 can be suppressed by gravity. Thereby, in the nucleic acid amplification reaction apparatus 200, the dispersion | variation in the brightness | luminance of fluorescence can be made small.

  In addition, when performing the fluorescence measurement of the reaction liquid 4 located in the 2nd temperature area | region 12b, compared with the case of performing the fluorescence measurement of the reaction liquid 4 located in the 1st temperature area | region 12a higher temperature than the 2nd temperature area | region 12b. It is possible to accurately measure fluorescence. For example, when the fluorescence measurement is performed using an intercalator type dye, the fluorescence measurement cannot be performed in a high temperature region such as the first temperature region 12a.

2.2. Second Modification Next, a nucleic acid amplification reaction apparatus according to a second modification of the present embodiment will be described with reference to the drawings. FIG. 5 is a cross-sectional view schematically showing a nucleic acid amplification reaction apparatus 300 according to the second modification of the present embodiment.

  In the nucleic acid amplification reaction apparatus 100 described above, as shown in FIG. 1, the moving mechanism 50 moves the support part 40 (reaction container 10, heating parts 20 and 22, and fluorescence measurement part 30) in the longitudinal direction L of the flow path 12. Move to. On the other hand, in the nucleic acid amplification reaction apparatus 300, as shown in FIG. 5, the moving mechanism 50 moves the support unit 40 in the direction N inclined with respect to the longitudinal direction L of the flow path 12. The direction N is a direction including the component of the longitudinal direction L. That is, the direction N is not a direction orthogonal to the longitudinal direction L. The inclination angle of the direction N with respect to the longitudinal direction L is not particularly limited as long as the reaction liquid 4 can be moved in the longitudinal direction L by moving the heating units 20 and 22 in the direction N. ° or less.

In the nucleic acid amplification reaction apparatus 300, the moving mechanism 50 moves the support unit 40 in the longitudinal direction L of the flow path 12.
Is moved in a direction N inclined with respect to. Therefore, in the nucleic acid amplification reaction device 300, the degree of freedom in the moving direction of the support unit 40 by the moving mechanism 50 can be increased.

  In the present invention, a part of the configuration may be omitted within a range having the characteristics and effects described in the present application, or each embodiment or modification may be combined.

  The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

DESCRIPTION OF SYMBOLS 4 ... Reaction liquid, 6 ... Liquid, 10 ... Reaction container, 12 ... Flow path, 12a ... 1st temperature range, 12b ... 2nd temperature range, 14 ... Bottom part, 16 ... Cover part, 20 ... 1st heating part, 21 ... 1st opening part, 22 ... 2nd heating part, 23 ... 2nd opening part, 30 ... Fluorescence measurement part, 40 ... Supporting part, 42 ... 1st part, 44 ... 2nd part, 46 ... 3rd part, 50 ... Movement mechanism, 60 ... Control unit, 100, 200, 300 ... Nucleic acid amplification reaction apparatus

Claims (9)

A mounting portion on which a reaction vessel in which a reaction solution is arranged in a flow path can be mounted;
A temperature gradient forming section for forming a temperature gradient in the longitudinal direction of the flow path;
A moving mechanism for moving the mounting portion in a direction including the component in the longitudinal direction and relatively moving the reaction solution in the longitudinal direction of the flow path;
A nucleic acid amplification reaction apparatus comprising: In claim 1,
The nucleic acid amplification reaction apparatus, wherein the moving mechanism swings the mounting portion. In claim 1 or 2,
The nucleic acid amplification reaction apparatus, wherein the moving mechanism moves the mounting portion in the longitudinal direction. In any one of Claims 1 thru | or 3,
The nucleic acid amplification reaction apparatus, wherein the longitudinal direction is a horizontal direction. In any one of Claims 1 thru | or 3,
The longitudinal direction is a direction inclined with respect to the horizontal direction,
The flow path is
A first temperature region and a second temperature region having a temperature lower than that of the first temperature region;
The nucleic acid amplification reaction apparatus, wherein the second temperature region is located below the first temperature region in a direction in which gravity acts. In any one of Claims 1 thru | or 5,
The nucleic acid amplification reaction apparatus, wherein the reaction container is filled with a liquid that is immiscible with the reaction liquid. In any one of Claims 1 thru | or 6,
The nucleic acid amplification reaction apparatus, wherein the moving mechanism changes the moving speed of the mounting portion in a direction including the longitudinal component to give acceleration to the reaction solution. In any one of Claims 1 thru | or 7,
The nucleic acid amplification reaction apparatus, wherein the moving mechanism imparts an acceleration larger than a gravitational acceleration to the reaction solution. Forming a temperature gradient in the longitudinal direction of the flow path in which the reaction solution is disposed;
Moving the reaction vessel having the flow path in a direction including the component in the longitudinal direction, and moving the reaction liquid relatively in the longitudinal direction of the flow path;
A nucleic acid amplification reaction method comprising:

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